Bioactivity guided isolation/ fractionation of anti-cancer principle from Nyctanthes arbor-tristis Linn

Abstract

Nyctanthes arbortristis Linn (Oleaceae) (NA) is one of the well-known medicinal plant in India. The parts of the plant contain many phyto compounds which have antioxidant, anti-inflammatory, antimalarial, expectorant, antileishmanial, immunomodulatory , anticancer and anthelmintic . There are no in-depth studies on the characterization of the exact bioactive phytoconstituent(s), which are present in the leaves of NA and responsible for their pharmacological effects. Aim of the study: 1.The aim of the study was to isolate the most bioactive compound(s) through bioactivity guided fractionation from NA leaves and also identify the bioactive compound(s) through various spectroscopic techniques such as FTIR, NMR and LC-MS. 2.Evaluation of anticancer activity of isolated compounds against various Human cancer cell lines – Hep G2 , A549 , HL-60 , MCF- 7 , HCT- 116 , PC- 3, HeLa – in vitro study . 3.Evaluation of anticancer, antioxidant activity of methanol leaf extract (MENA) on EAC cells. 4. Investigation of biochemical parameters and histological architecture of liver in various groups of Swiss Albino mice . Material and Methods: The methanol, ethyl acetate, n-butanol and aqueous fractions of NA leaves were prepared and tested for their anti-inflammatory, anticancer and antioxidant activity in both cell-free (COX-1, COX-2, 5-LOX and DPPH) and cell-based assays (NO, TNF-α in RAW264.7 and cytotoxicity in HepG2). The most bioactive fraction was further separated, purified using column chromatography; isolated and identified the bioactive compound using FTIR, NMR and mass spectroscopy. Antitumor and anticancer activity of MENA were investigated on Swiss Albino mice against EAC cells (In vivo study). Results: The highest anti-inflammatory, anticancer and antioxidant activity were found in ethyl acetate fraction in both cell-free and cell-based assays. IC50 values of ethyl acetate fractions were 160.56 – 180.44 μg/mL for anti-inflammatory, 145.48 μg/mL for anticancer, and 190.75 μg/mL for antioxidant activity. Therefore, ethyl acetate fraction was used for further separation, purification using column chromatography; isolated and identified as Betulinic acid through spectral analysis. Betulinic acid had shown excellent inhibition of anti-inflammatory properties with IC50 of 10.34 μg/mL (COX-1), 12.92 μg/mL (COX-2), 15.53 μg/mL (5-LOX), 15.21 μg/mL (Nitrite), 16.65 μg/mL (TNF-α), and also exhibited potent anticancer and IC50 of 18.03 μg/mL and 6.53 -17.07 μM respectively. In-vivo study of MENA showed the increased level of CAT, SOD, GSH and reduced level of LPO compared to EAC control group of mice. Biochemical parameters SGPT, SGOT, Total Protein, Bilirubin, Haemoglobin, RBC, WBC counts were normalized. Life Span of the EAC inoculated and MENA treated group of mice were increased. Conclusion: This is the first report on the isolation of betulic acid, lupane-type triterpenoid via bioactivity-guided fractionation from leaves of Nyctanthes arbor-tristis. Overall, the bioactivity results suggest that betulinic acid has a great potential to consider a suitable candidate for the drug development for its anti-inflammatory, anticancer and antioxidant activity.